Apparatus for forming molecular timetable and apparatus for estimating circadian clock

ABSTRACT

An apparatus  11  for estimating internal biological time of a biological individual on the basis of quantity data of a gene product as measured in a specimen sampled from the individual. The apparatus includes circadian oscillatory gene selection means for selecting circadian oscillatory genes whose time-course change in gene product quantity data as measured in standard specimens approximates a cosine curve having a predetermined period; circadian expression curve selection means for selecting, from among a plurality of cosine curves having different phases and sharing a specific period, a circadian expression curve which is similar to the pattern of time-course change in the expression product quantity of each of the above-selected circadian oscillatory genes; and registration means for registering information which identifies the selected circadian expression curve.

TECHNICAL FIELD

The present invention relates to a molecular timetable generatingapparatus, an internal biological time estimating apparatus, a moleculartimetable generation method, an internal biological time estimatingmethod, a molecular timetable generating program, an internal biologicaltime estimating program, and an internal biological time estimatingsystem. In particular, the invention relates to a molecular timetablegenerating apparatus, an internal biological time estimating apparatus,a molecular timetable generation method, an internal biological timeestimating method, a molecular timetable generating program, an internalbiological time estimating program, and an internal biological timeestimating system; the apparatuses, methods, programs, and the systemfacilitating estimation of internal biological time, by use of acomputer, from results of a test performed on a specimen obtained fromsingle-time-point sampling.

BACKGROUND ART

Many organisms have internal biological clocks, which control variousbiological rhythms such as sleep and wakefulness rhythm, blood-pressurerhythm, body-temperature rhythm, and some hormonal secretion rhythms.

The oscillation cycle of internal biological clocks is approximately 24hours, but the cycle slightly differs from biological species tospecies. In the case of humans, the oscillation cycle is said to beabout 25 hours. A rhythm of around 24 hours in a steady environmentwhich involves no cyclic change is called “circadian rhythm.” In dailylife, sunlight serves as the strongest synchronization factor forsynchronizing, day by day, the circadian rhythm with a 24-hour cycle.

An anomaly in circadian rhythm is a known cause of sleep-wake rhythmdisorders (e.g., delayed sleep phase syndrome, advanced sleep phasesyndrome, non-24-hour sleep-wake rhythm syndrome), seasonal depression,jet lag syndrome, sleep disturbance experienced by day/night shiftworkers, nighttime wandering of aged persons suffering dementia, andother pathological conditions such as delirium, as well as a cause ofschool truancy or refusal to go to work.

Moreover, the time of day a person takes a medicine (medicine intaketime) is known to affect the efficacy of the medicine, strength of itsside effects, and pharmacokinetics. The internal biological time of anight worker, a shift worker, or the like is sometimes different fromthat of ordinary persons. Therefore, when such a person takes a medicineat a timing suitable for ordinary persons, the medicine is not expectedto provide a sufficient effect unless the timing matches his circadiantime suitable for taking the medicine.

Accordingly, for the purpose of, for example, diagnosis of personssuffering circadian rhythm disorders, determination of medicine intaketimes on an individual basis, or prevention of nighttime wandering ofaged persons suffering dementia, there has been demand for developmentof a system capable of determining an individual's internal biologicaltime on an individual basis and offering information on the individual'sinternal biological time to the neurological departments of medicalfacilities, medical doctors who prescribe medicines, hospitals for theelderly, and health care facilities for the elderly. Thus, research on amethod for estimating internal biological time is being developed.

Conceivable methods for estimating internal biological time include amethod in which blood is drawn from an individual over time to therebymeasure the blood melatonin level, and the individual's internalbiological time is estimated on the basis of the peak melatonin leveland the time at which the peak level is obtained; a method in whichactivity level and sleep of an individual is measured over time fordetermining the sleep-wake rhythm of the individual, whereby theindividual's internal biological time is estimated; and a method inwhich the body temperature of an individual is measured so as todetermine the body temperature rhythm of the individual, whereby theindividual's internal biological time is estimated.

However, these methods have a drawback in that drawing of blood,body-temperature measurement, or the like must be performed as timeelapses over 24 hours or more, which imposes a great burden on bothpatients and medical facilities. Therefore, these are not practicalmethods suitable for use in actual medical sites. Meanwhile, in somework related to time-course measurement of the quantity of gene productsof drosophila heads by use of oligo-DNA arrays (McDonald, M. andRosbash, M., Cell, 107, 567-578. (2001), Claridge-Chang, A. et al.,Neuron, 32, 657-671(2001)) or to investigation of circadian oscillatorygenes in mouse liver by use of the differential display method(Kornmann, B. et al., Nucleic Acids Res., 29(11), e51 (2001)), manycircadian oscillatory genes have been identified in relevant tissues,and in addition, times at which respective circadian oscillatory genesexhibit the maximum expression levels have also been reported.

However, no method for determining internal biological time by use ofdata obtained from existing methods as described above is known, andthere still remains demand for establishment of a method for measuringinternal biological time.

An object of the present invention is to solve the above problem byprovision of a molecular timetable generating apparatus, an internalbiological time estimating apparatus, a molecular timetable generationmethod, an internal biological time estimating method, a moleculartimetable generating program, an internal biological time estimatingprogram, and an internal biological time estimating system; theapparatuses, methods, programs, and the system facilitating estimationof an individual's internal biological time through one drawing of aspecimen; i.e., without need for a plurality of times of specimencollection.

Another object of the present invention is to provide a moleculartimetable generating apparatus, an internal biological time estimatingapparatus, a molecular timetable generation method, an internalbiological time estimating method, a molecular timetable generatingprogram, an internal biological time estimating program, and an internalbiological time estimating system; the apparatuses, methods, programs,and the system facilitating estimation of an individual's internalbiological time enabling provision of results of estimation of anindividual's internal biological time to a client such as a medicalfacility, a health care facility for the elderly, and a sports club.

DISCLOSURE OF THE INVENTION

As defined in claim 1 of the present invention, the molecular timetablegenerating apparatus of the present invention is an apparatus forgenerating a molecular timetable for estimating internal biological timeof a biological individual on the basis of quantity data of a geneproduct (hereinafter referred to as gene product quantity data) asmeasured in standard specimens sampled from individuals, the apparatuscomprising: data input means for inputting gene product quantity data ofstandard specimens each sampled from a predetermined portion of each ofa plurality of individuals of a predetermined biological species;circadian oscillatory gene selection means for selecting, from among thegenes which are expressed in the aforementioned standard specimens,circadian oscillatory genes whose time-course change in gene productquantity data approximates a cosine curve having a predetermined period;circadian expression curve selection means for selecting, from among aplurality of cosine curves having different phases and sharing aspecific period, a circadian expression curve which is similar to thepattern of time-course change in the expression product quantity of eachof the above-selected circadian oscillatory genes; and registrationmeans for registering information which identifies the selectedcircadian expression curve.

Some of the above-mentioned objects are attained by a moleculartimetable generating method as defined in claim 4, which enables, by useof information processing equipment, generation of a molecular timetablefor estimating internal biological time of a biological individual onthe basis of gene product quantity data as measured in standardspecimens sampled from individuals, the method comprising: a data inputprocedure for inputting gene product quantity data of standard specimenseach sampled from a predetermined portion of each of a plurality ofindividuals of a predetermined biological species; a circadianoscillatory gene selection procedure for selecting, from among the geneswhich are expressed in the aforementioned standard specimens, circadianoscillatory genes whose time-course change in gene product quantity dataapproximates a cosine curve having a predetermined period; a circadianexpression curve selection procedure for selecting, from among aplurality of cosine curves having different phases and sharing aspecific period, a circadian expression curve which is similar to thepattern of time-course change in the expression product quantity of eachof the above-selected circadian oscillatory genes; and a registrationprocedure for registering information which identifies the selectedcircadian expression curve.

Some of the above-mentioned objects are attained by a moleculartimetable generating program as defined in claim 6, which enablesinformation processing equipment to perform: a data input procedure forinputting gene product quantity data of standard specimens each sampledfrom a predetermined portion of each of a plurality of individuals of apredetermined biological species; a circadian oscillatory gene selectionprocedure for selecting, from among the genes which are expressed in theaforementioned standard specimens, circadian oscillatory genes whosetime-course change in gene product quantity data approximates a cosinecurve having a predetermined period; a circadian expression curveselection procedure for selecting, from among a plurality of cosinecurves having different phases and sharing a specific period, acircadian expression curve which is similar to the pattern oftime-course change in the expression product quantity of each of theabove-selected circadian oscillatory genes; and a registration procedurefor registering information which identifies the selected circadianexpression curve; said information processing equipment being providedfor generation of a molecular timetable for estimating internalbiological time of the biological individual on the basis of geneproduct quantity data as measured in standard specimens sampled fromindividuals.

Some of the above-mentioned objects are attained by an internalbiological time estimation system as defined in claim 8 for enablinggeneration of a molecular timetable for estimating internal biologicaltime of a biological individual on the basis of gene product quantitydata as measured in standard specimens sampled from individuals andestimation of internal biological time of the biological individual onthe basis of gene product quantity data as measured in a specimensampled from the biological individual; said system comprising: a servercomputer installed in an information center for providing internalbiological time information, and a terminal computer connected to theserver computer so as to transmit and receive information therebetween,wherein the server computer includes: standard data input means forinputting gene product quantity data of standard specimens each sampledfrom a predetermined portion of each of a plurality of individuals of apredetermined biological species; circadian oscillatory gene selectionmeans for selecting, from among the genes which are expressed in theaforementioned standard specimens, circadian oscillatory genes whosetime-course change in gene product quantity data approximates a cosinecurve having a predetermined period; circadian expression curveselection means for selecting, from among a plurality of cosine curveshaving different phases and sharing a specific period, a circadianexpression curve which is similar to the pattern of time-course changein the expression product quantity of each of the above-selectedcircadian oscillatory genes; registration means for registering, as astandard molecular time of the circadian oscillatory gene, a point intime at which the circadian expression curve reaches the maximum, in themolecular timetable used for estimating the internal biological time,and also for registering, in the molecular timetable, an average valueand standard deviation, both calculated for each circadian oscillatorygene, of the expression product quantity data, as a standard expressionquantity and standard variation of the circadian oscillatory gene,respectively; measurement data input means for inputting the geneproduct quantity data of the circadian oscillatory genes contained inthe specimen sampled from the said predetermined portion of thebiological individual; internal biological time derivation means forobtaining internal biological time information including judgment as towhether or not the biological individual suffers a circadian rhythmdisorder, and when the biological individual does not suffer a circadianrhythm disorder, an estimated internal biological time of the biologicalindividual, through comparison between the input measurement data andthe circadian expression curve specified by the molecular timetable; andinternal biological time transmission means for transmitting theobtained internal biological time information to the terminal computer.

Thus, the present invention enables generation of timetables each ofwhich specifies a circadian expression curve of a circadian oscillatorygene contained in a predetermined portion of a predetermined biologicalspecies, due to the provision of circadian oscillatory gene selectionmeans for selecting, from among the genes which are expressed in theaforementioned standard specimens, circadian oscillatory genes whosetime-course change in gene product quantity data approximates a cosinecurve having a predetermined period; circadian expression curveselection means for selecting, from among a plurality of cosine curveshaving different phases and sharing a specific period, a circadianexpression curve which is similar to the pattern of time-course changein the expression product quantity of each of the above-selectedcircadian oscillatory genes; and registration means for registeringinformation which identifies the selected circadian expression curve. Inother words, the invention enables provision, for an individualbelonging to the biological species, of a timetable which serves as thebasis for determining as to whether or not that individual has acircadian rhythm disorder, and when the individual does not have acircadian rhythm disorder, estimating the internal biological time ofthat individual.

Preferably, the registration means is configured so as to register, as astandard molecular time of the circadian oscillatory gene, a point intime at which the circadian expression curve reaches the maximum, in themolecular timetable used for estimating the internal biological time,and also to register, in the molecular timetable, an average value andstandard deviation, both calculated for each circadian oscillatory gene,of the expression product quantity data, as a standard expressionquantity and standard variation of the circadian oscillatory gene,respectively.

With this configuration, the present invention enables generation of amolecular timetable which specifies a circadian expression curve of acircadian oscillatory gene contained in a predetermined portion of apredetermined biological species. In other words, the invention enablesprovision, for an individual belonging to the biological species, of amolecular timetable which may be used for determining as to whether ornot that individual has a circadian rhythm disorder, and when theindividual does not have a circadian rhythm disorder, estimating theinternal biological time of that individual. Moreover, registration of acircadian expression curve can be attained by use of the following dataonly; i.e., standard molecular time, standard expression level, andstandard variation. Thus, circadian expression curves can be managed andcontrolled using a smaller number of data.

Some of the above-mentioned objects are attained by an internalbiological time estimation apparatus as defined in claim 3 forestimating internal biological time of a biological individual on thebasis of gene product quantity data as measured in specimens sampledfrom individuals, the apparatus comprising: molecular timetable storagemeans for storing molecular timetables each of which specifies acircadian expression curve showing time-course change in the expressionproduct quantity of a circadian oscillatory gene contained in apredetermined portion of a predetermined biological species; data inputmeans for inputting the gene product quantity data of the circadianoscillatory genes contained in a specimen sampled from saidpredetermined portion of the biological individual; and internalbiological time derivation means for obtaining internal biological timeinformation including judgment as to whether or not the biologicalindividual suffers a circadian rhythm disorder, and when the biologicalindividual does not suffer a circadian rhythm disorder, an estimatedinternal biological time of the biological individual, throughcomparison between the input measurement data and the circadianexpression curve specified by the molecular timetable.

Some of the above-mentioned objects are attained by an internalbiological time estimation method as defined in claim 5 for estimating,by use of information processing equipment, internal biological time ofa biological individual on the basis of gene product quantity data asmeasured in specimens sampled from individuals, the method comprising: amolecular timetable storage procedure for storing molecular timetableseach of which specifies a circadian expression curve showing time-coursechange in the expression product quantity of a circadian oscillatorygene contained in a predetermined portion of a predetermined biologicalspecies; a data input procedure for inputting the gene product quantitydata of the circadian oscillatory genes contained in a specimen sampledfrom said predetermined portion of the biological individual; and aninternal biological time derivation procedure for obtaining internalbiological time information including judgment as to whether or not thebiological individual suffers a circadian rhythm disorder, and when thebiological individual does not suffer a circadian rhythm disorder, anestimated internal biological time of the biological individual, throughcomparison between the input measurement data and the circadianexpression curve specified by the molecular timetable.

Some of the above-mentioned objects are attained by an internalbiological time estimation program as defined in claim 7, which enablesinformation processing equipment to perform: a molecular timetablestorage procedure for storing molecular timetables each of whichspecifies a circadian expression curve showing time-course change in theexpression product quantity of a circadian oscillatory gene contained ina predetermined portion of a predetermined biological species; a datainput procedure for inputting the gene product quantity data of thecircadian oscillatory genes contained in a specimen sampled from saidpredetermined portion of the biological individual; and an internalbiological time derivation procedure for obtaining internal biologicaltime information including judgment as to whether or not the biologicalindividual suffers a circadian rhythm disorder, and when the biologicalindividual does not suffer a circadian rhythm disorder, an estimatedinternal biological time of the biological individual, throughcomparison between the input measurement data and the circadianexpression curve specified by the molecular timetable; said informationprocessing equipment being provided for estimating internal biologicaltime of the biological individual on the basis of gene product quantitydata as measured in standard specimens sampled from individuals.

As described above, with an internal biological time derivation meansfor obtaining internal biological time information including judgment asto whether or not the biological individual suffers a circadian rhythmdisorder, and when the biological individual does not suffer a circadianrhythm disorder, an estimated internal biological time of the biologicalindividual, through comparison between the input measurement data andthe circadian expression curve specified by the molecular timetable, acomparison of assay data on a specimen sampled at a single point in timewith the above-mentioned circadian expression curves specified bymolecular timetables generated for a predetermined portion of apredetermined biological species achieves, without need for samplingspecimens a plurality of times, a simple judgment procedure fordetermining, on the basis of a specimen sampled at a single point intime, as to the presence or absence of a circadian rhythm disorder, andfor estimating the internal biological time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an explanatory diagram showing a general configuration of theinternal biological time estimation system according to one embodimentof the present invention;

FIG. 2 is an explanatory diagram showing a hardware configuration of theinternal biological time estimation apparatus for general management ofthe internal biological time estimation system according to oneembodiment of the present invention;

FIG. 3 is an explanatory diagram showing the makeup of a time-coursegene product quantity table;

FIG. 4 is an explanatory diagram showing the makeup of a moleculartimetable;

FIG. 5 is an explanatory diagram showing the makeup of an internalbiological time information table;

FIG. 6 is a block diagram showing a process flow of the internalbiological time estimation system according to one embodiment of thepresent invention;

FIG. 7 is a flowchart showing the circadian oscillation gene selectionprocess according to one embodiment of the present invention;

FIG. 8 an explanatory diagram showing cosine waves for gene selection;

FIG. 9 is a flowchart showing a process for creating circadianexpression curves and molecular timetables according to one embodimentof the present invention;

FIG. 10 is a flowchart showing an estimation process for internalbiological time and information provision process according to oneembodiment of the present invention;

FIG. 11 is a flowchart showing an initial setting process, which is partof the estimation process for internal biological time and informationprovision process according to one embodiment of the present invention;

FIG. 12 is a flowchart showing a normalization process for circadianoscillation gene product quantity of a sample, the normalization processbeing part of the estimation process for internal biological time andinformation provision process according to one embodiment of the presentinvention;

FIG. 13 is a flowchart showing a calculation process of the Pearson'scorrelation coefficient c between a normalized circadian oscillationgene product quantity and an estimated expression level for each ofcircadian oscillation genes at time t, the calculation process beingpart of the estimation process for internal biological time andinformation provision process according to one embodiment of the presentinvention;

FIG. 14 is an explanatory diagram showing an internal biological timeinformation report screen according to one embodiment of the presentinvention;

FIG. 15 is an explanatory diagram showing a test subject characteristicstable; and

FIG. 16 is an explanatory diagram showing exemplary data analysisresults.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will next be described withreference to the drawings. The elements, configurations, etc. describedhereinbelow should not be construed as limiting the present invention,and various modifications can be made within the scope of the invention.

The internal biological time estimating system of the present inventionderives, from the gene product quantity data of a human specimen inputin or received by the server computer in an information center, internalbiological time information including a judgment as to whether or notthe specimen donor suffers a circadian rhythm disorder, and in the casewhere the specimen donor is determined not to have a circadian rhythmdisorder, an estimated internal biological time of the specimen donor,and then transmits the internal biological time information to aterminal computer of the client who requested the test.

According to the present embodiment, internal biological time of a humanis estimated, but the internal biological time estimating system of thepresent embodiment may be employed for estimating internal biologicaltime of any living organism so long as the organism has a circadianrhythm, and examples include mammals such as mice, rats, and dogs.

Also, the present embodiment will be described in relation to a casewhere internal biological time of an individual is estimated by use of ahuman blood specimen, but any specimen may be employed for estimatinginternal biological time, so long as the specimen is originating from anorganism having a circadian rhythm. Examples of such a specimen includethe suprachiasmatic nucleus or the liver of a mouse, drosophila heads,human skin, white blood cells from peripheral blood, mucous membrane ofthe oral cavity, and other organs.

Further, according to the present embodiment, quantity data for a genetranscription product, messenger RNA (hereinafter referred to as mRNA),are employed as the gene product quantity data, but any measurement datamay be employed, so long as the data exhibit a circadian rhythm.Examples of the measurement data include those of the mass of a proteincoded by a gene, those of modification levels of a protein coded by agene, those of activity levels of an enzyme coded by a gene, those of acompound metabolized by an enzyme, and those related to physical itemsregulated by gene products, such as body temperature, blood pressure,heart rate, blood flow, respiration rate, brain waves, activity level,and pH of a body fluid.

The term “circadian oscillatory gene” denotes a gene which isoscillatorily expressed with a cycle period of about 24 hours (20 to 28hours) under light/dark conditions (i.e., alternating 12-hour lightperiod/12-hour dark period) or constant dark conditions.

The term “standard expression level” denotes a mean value of time-courseexpression level measurement data of a gene; “standard variation”denotes standard deviation of time-course expression level measurementdata of the gene; and “relative expression level” denotes an expressionlevel normalized by subtracting the standard expression level from ameasurement value in items of a gene product of a gene, followed bydivision by the standard variation.

Normalization is means for assessing expression variation of each geneon the same basis, and specifically, the term “normalized” or“normalization” denotes a process of subtracting the standard expressionlevel from the measured value for the quantity of a gene product of eachgene, or a value extracted from a corresponding circadian expressioncurve, followed by division by the standard variation. Through thisnormalization process, the standard variation and the standardexpression level of each circadian oscillatory gene become 1 and 0,respectively, resulting in unified treatment of these variables.

A “circadian expression curve” is drawn from a numerical formula oftime-course expression profile of a circadian oscillatory gene derivedfrom a finite number of expression data. The curve can be obtainedthrough any of a plurality of methods on the basis of expression levelsmeasured over time. A “relative circadian expression curve” is anormalized circadian expression curve obtained by subtracting thestandard expression level from a value extracted form a correspondingcircadian expression curve, followed by division by the standardvariation.

A “ZT time” is a time measured from when a light is turned on (ZT0)under the light/dark conditions. The light remains on for 12 hours, andthen the light is turned off at ZT12. Thus, the light period is from ZT0to ZT12, and the dark period is from ZT12 to ZT24.

A “CT time” is a time determined on a subject which has undergone rhythmsynchronization, or entrainment, to a 24-hour light/dark period. Thebeginning of the CT time, CT0, corresponds to ZT0, but under the CTtimescale, the light is not turned on at CT0. The CT time is measuredunder constant dark conditions and has a period of 24 hours.

A “molecular time” is a time on a CT timescale (CT time) or a time on aZT timescale (ZT time), at which the circadian expression curve of thecircadian oscillatory gene exhibits the maximum value.

Relations between objects which are “similar” to one another areexpressed by numerical values. Such relations include “similarity,”which is used when, as in the case of correlation coefficient, a largervalue represents a more similar relation, and “dissimilarity,” which isused when, as in the case of distance, a smaller value represents a lesssimilar relation.

The “distance,” one of the indices representing dissimilarity, is anindex showing how far the objects are separated from one another. The“squared Euclidian distance” is one of the most commonly used distance,and the distance d_(xy) between variables x and y is defined as followswhen the data are given in the form of (x₁, y₁), (x₂, y₂), (x₃, y₃), . .. , (x_(n), y_(n)) (see Reference 3). $\begin{matrix}{d_{xy} = {\sum\limits_{i = 1}^{N}\quad\left( {x_{i} - y_{i}} \right)^{2}}} & (1)\end{matrix}$

The “correlation coefficient,” one of the indices representingsimilarity, shows the degree of correlation. The “Pearson's productmoment correlation coefficient” is one of the most commonly usedcorrelation coefficients and is defined as follows (see Reference 4).$\begin{matrix}{{\gamma_{xy} = \frac{\sum\limits_{i = 1}^{N}\quad\frac{\left( {x_{i} - \overset{\_}{x}} \right)\left( {y_{i} - \overset{\_}{y}} \right)}{n}}{\sqrt{\sum\limits_{i = 1}^{N}\quad\frac{\left( {x_{i} - \overset{\_}{x}} \right)^{2}}{n}}\sqrt{\sum\limits_{i = 1}^{N}\quad\frac{\left( {y_{i} - \overset{\_}{y}} \right)^{2}}{n}}}}{where}} & (2) \\{{\overset{\_}{x} \equiv \frac{\sum\limits_{i = 1}^{N}\quad x_{i}}{n}},{\overset{\_}{y} \equiv \frac{\sum\limits_{i = 1}^{N}\quad y_{i}}{n}}} & (3)\end{matrix}$

Next, the standard deviation s of a cosine wave will be described. Thevalue A(t) of the cosine wave at time t can be obtained from:$\begin{matrix}{{A(t)} = {{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{Period} \right)}} & (4)\end{matrix}$where

-   Period: 24 hours,-   T: the molecular time on the cosine wave (e.g., 144 kinds of times    at 10-minute intervals; i.e., 0/60, 10/60, 20/60, 30/60, . . . ,    1420/60, and 1430/60), and-   t: corresponding time.

The standard deviation s of the time-course data (a set of values atrelevant sampling points in time) on the cosine wave can be obtainedfrom the following formula. $\begin{matrix}{s = \sqrt{\frac{{n{\sum\limits_{i = 1}^{N}\quad\left( {A(t)}_{i} \right)^{2}}} - \left( {\sum\limits_{i = 1}^{n}\quad{\left( {A(t)} \right)i}} \right)^{2}}{n\left( {n - 1} \right)}}} & (5)\end{matrix}$

If the time-course data on the cosine wave are obtained at intervals of4 hours, t can be expressed by the following set of the 6 time points(n=6): (α+0)/60, (α+240)/60, (α+480)/60, (α+720)/60, (α+960)/60, and(α+1200)/60, where α is a certain time between 0 and 240 minutes(exclusive of 240 minutes). In this case, the standard deviation is0.7745967.

In the present specification, the term “computer” is used to mean anyinformation terminal equipped with an arithmetic unit. Examples of thecomputers include a supercomputer, a general purpose computer, an officecomputer, a computer for control purposes, a workstation, and a personalcomputer, and further include a cellular information terminal, acellular phone equipped with an arithmetic unit, and a wearablecomputer.

Also, as used herein, the term “input” means inputting an instruction ordata to a computer, and includes inputting an instruction or datathrough a keyboard or a mouse, reading an instruction or data from astorage medium such as a flexible disk or a compact disk, and receivingan instruction or data from an external unit through a communicationline.

EMBODIMENT 1

As shown in FIG. 1, the internal biological time estimating systemaccording to this embodiment of the present invention includes aninformation center 1 providing internal biological time information ofan individual to a client and clients such as a medical facility 2, asports center 3, an individual member 4, a health care facility for theelderly 5, a corporate member 6, and an educational institution 7. Theseclients receives, from the information center 1, internal biologicaltime information of an individual.

The information center 1 has a server computer 11, and terminalcomputers 21 to 71 belonging to clients 2 to 7 are connected thereto viathe Internet 13.

The information center 1 is a general administration organization foroperating the internal biological time estimating system of the presentembodiment, and may be included in a clinical test company, apharmaceutical company, a medical facility, a research institute, ananalysis center, an examination center, a data center, or the like.

The information center 1 includes a DNA chip reader 12 for measuring thequantity of a gene product of a specimen and inputting the gene productquantity data to the server computer 11; and the server computer 11,serving as an internal biological time information providing unit, forperforming, by use of the gene product quantity data transmitted fromthe DNA chip reader 12, processes related to judgment on the presence orabsence of a circadian rhythm disorder or to estimation of internalbiological time, and subsequently transmitting to any of the terminalcomputers 21 to 71 the internal biological time information obtainedthrough the processes.

In the present embodiment, the gene product quantity data are sent fromthe DNA chip reader 12. Alternatively, an apparatus for measuring thegene product quantity may be installed in each of the clients 2 to 7 forsending the data from the terminal computers 21 to 71.

The information center 1 receives, from the clients 2 to 7, by mail orwalk-in, blood samples 19 drawn from subjects undergoing clinical tests(hereinafter may be referred to as simply test subjects or subjects).Each blood sample 19 is pretreated through a known method, to therebymake the sample ready for measurement of expressed mRNA level of a gene.For example, in one known method, mRNA is separated/purified from theblood sample 19 by use of a commercial RNA extraction kit (e.g.,RNAqueous-Blood Module, RNAqueous Phenol-free Total RNA kit, FunakoshiCo., Ltd.) with reference to the protocol provided by the manufacturer.Purification of the blood sample 19 may be performed by extracting mRNAby use of, for example, the guanidine thiocyanate/cesium chloridemethod, the guanidine thiocyanate hot phenol method, or the guanidinethiocyanate-guanidine hydrochloride method, and then adsorbing/elutingmRNA on an oligo(dT)-cellulose column.

Subsequently, the quantity of expressed mRNA in the treated blood sample19 is measured by the DNA chip reader 12. The thus-obtained raw mRNAquantity data are accumulated in the DNA chip reader 12.

The raw data accumulated in the DNA chip reader 12 are then transmittedto the server computer 11. The server computer 11 processes the rawdata, determines whether the relevant test subject has circadian rhythmdisorders, and, if the subject does not have circadian rhythm disorders,calculates the internal biological time of the subject.

The results of judgment as to whether or not the test subject has acircadian rhythm disorder and, in the case where the subject does nothave a circadian rhythm disorder, the calculated internal biologicaltime of the test subject are transmitted and reported, as the biologicaltime information of the test subject, to the relevant terminal computer;i.e., the relevant one of the terminal computers 21 to 71, belonging tothe client who requested the test.

The clients 2 to 7 are clients of the information center 1 and receivethe internal biological time information produced by the internalbiological time estimating system of the present embodiment at theinformation center 1.

Each of the clients 2 to 7 has a terminal computer; i.e., a relevant oneof the terminal computers 21 to 71, which enables the client to view thedisplay internal biological time information display screen provided bythe server computer 1 at the information center 1.

A gene product quantity measuring apparatus for measuring the quantityof a gene product may be installed in each of the clients 2 to 7. Insuch a case, the expression level of mRNA of a blood sample 19 of a testsubject is measured at any of the clients 2 to 7, and the obtained dataare transmitted to the server computer 11 from the corresponding one ofthe terminal computers 21 to 71, and subsequently the same one of theterminal computers 21 to 71 receives, from the server computer 11, thejudgment as to whether or not the data indicate the presence of acircadian rhythm disorder and the internal biological time informationderived from the data.

The medical facility 2 utilizes the internal biological time estimatingsystem of the present embodiment for purposes of, among other purposes,determining whether or not there is involved a circadian rhythmdisorders in a patient suffering a sleep-wake rhythm disorder, seasonaldepression, or jet-time syndrome or, in the case where the patient is aday-night shift worker, suffering a sleep disorder, or determining thetime at which a patient should take a medicine.

The medical facility 2 has an electronic medical record server computer22 for storing electronic data on medical treatment records,prescriptions, clinical test results, and other information pertainingto individual patients. The electronic medical record server computer 22is connected to a terminal computer 21 via an intra-hospital LAN, and isconfigured such that internal biological time information downloadedfrom the server computer 11 to the terminal computer 21 can be input tothe electronic medical record server computer 22.

The electronic medical record server computer 22 may be configured suchthat internal biological time information pertaining to a patient can beautomatically and directly incorporated to his/her electronic medicalrecord. This can be realized by allotting to each patient, as his/hertest subject number (described hereinbelow), his/her patient ID numberregistered in the electronic medical record server computer 22, and byinstalling a program which automatically transfers, to a medical recorddatabase (not shown) provided in the electronic medical record servercomputer 22, the internal biological time information pertaining to eachtest subject downloaded from the server computer 11 to the terminalcomputer 21.

The medical facility 2 may be replaced by, for example, a psychologicalcounselor, a facility for health and welfare such as a health center, aclinic, a child counseling facility, or a pharmacy.

The sports center 3 is an organization in which athletes are coached,trained, or nurtured. No particular limitation is imposed on the typesof the sports. In the sports center 3, the internal biological timeestimating system of the present embodiment is used to establishtraining schedules for athletes who are scheduled to take part ininternational sports events.

The sports center 3 has, in addition to a terminal computer 31, anathlete management server computer 32 which stores electronic data, foreach athlete, concerning, for example, health conditions, a trainingmenu, a diet menu, or event participation records pertaining to anathlete. The athlete management server computer 32 is connected to theterminal computer 31 via an intra-center LAN, and is configured suchthat internal biological time information downloaded from the servercomputer 11 to the terminal computer 31 can be input to the athletemanagement server computer 32.

The athlete management server computer 32 may be configured such thatinternal biological time information pertaining to each athlete can beautomatically and directly incorporated to a database (not shown)provided in the athlete management server computer 32. This can berealized by allotting to each athlete, as his/her test subject number(described hereinbelow), his/her athlete ID number registered in theathlete management server computer 32, and by installing a program whichautomatically transfers, to the unillustrated database, the internalbiological time information pertaining to the athlete downloaded fromthe server computer 11 to the terminal computer 31.

The sports center 3 may be replaced by a sports gym, a sports club, alegal person who manages club activities, or an educational institutionwhich governs club activities.

The individual member 4 is a client who privately utilizes the internalbiological time estimating system of the present embodiment for thepurpose of improving his/her own life.

Examples of the individual member 4 include a health-consciousindividual, a senior citizen, a person who has experienced to suffer asleep-wake rhythm disorder, seasonal depression, jet-lag syndrome, or asimilar disorder, and a day-night shift worker. When a family member ofan individual member 4 or a person who cares for an individual member 4can draw a specimen from the individual member, the family member or theperson, instead of the individual member 4 himself/herself, may draw aspecimen and view or handle the internal biological time information ofthat individual.

The health care facility for the elderly 5 makes use of the internalbiological time estimating system of the present embodiment for purposesof health management and daily life management of the elderly staying inthe facility.

For example, internal biological time information may be used asinformation for allotting rooms to potential inmates.

When an aged person moves in such a facility, the facility acquires hisor her internal biological time information from the information center1, whereby inmates having similar internal biological time patterns canbe grouped to share a room. This facilitates administration andmanagement over inmates (e.g., patrol at wake-up time and at bedtime),as the inmates in the same room have similar internal biological timeand thus can live on similar behavioral paces.

In addition, if internal biological time information of an aged personis acquired at the time when he or she moves in the facility, the staffof the facility can adequately respond to his or her daily life rhythmfrom an early stage after his or her relocation, facilitating adaptationof the new corner to the general timetable of the facility from an earlytime.

The health care facility for the elderly 5 has, in addition to aterminal computer 51, a management server computer 52 which storeselectronic data, for each inmate, concerning health conditions, a dietmenu, information about family members, a rehabilitation menu, or otherinformation pertaining to an inmate. The management server computer 52is connected to the terminal computer 51 via an intra-facility LAN, andis configured such that internal biological time information downloadedfrom the server computer 11 to the terminal computer 51 can be inputinto the management server computer 52.

The management server computer 52 may be configured such that internalbiological time information pertaining to each inmate can beautomatically and directly incorporated to a database (not shown)provided in the management server computer 52. This can be realized byallotting to each inmate, as his/her test subject number (describedhereinbelow), his/her inmate ID number registered in the managementserver computer 52, and by installing a program which automaticallytransfers, to the unillustrated database, the internal biological timeinformation pertaining to the inmate downloaded from the server computer11 to the terminal computer 51.

Other than the health care facility for the elderly 5, example clientsinclude hospitals for the elderly, senior welfare facilities, nursinghomes, and companies providing services for supporting family memberswho care for aged persons.

The corporate member 6 utilizes the internal biological time estimatingsystem of the present embodiment for purposes of, for example,management of employees' health conditions and improvement of employees'work performance.

The corporate member 6 has, in addition to a terminal computer 61, anemployee information server computer 62 which stores electronic data,for each employee, concerning, for example, working hours, working timezone, jobs or tasks, health conditions, and the age of the employee. Theemployee information server computer 62 is connected to the terminalcomputer 61 via a LAN, and is configured such that internal biologicaltime information downloaded from the server computer 11 to the terminalcomputer 61 can be input to the employee information server computer 62.

The employee information server computer 62 may be configured such thatinternal biological time information pertaining to each employee can beautomatically and directly incorporated to the employee information.This can be realized by allotting to each employee, as his/her testsubject number (described hereinbelow), his/her employee ID numberregistered in the employee information server computer 62, and byinstalling a program which automatically transfers, to an unillustrateddatabase, the internal biological time information pertaining to theemployee downloaded from the server computer 11 to the terminal computer61.

The corporate member 6 may be replaced by, for example, a public agencyor a domestic or abroad branch office thereof.

The educational institution 7 utilizes the internal biological timeestimating system of the present embodiment for purposes of, among otherpurposes, management of physical or mental health of pupils and studentsor improvement of education efficiency.

The educational institution 7 has, in addition to a terminal computer71, a pupil or student information server computer 72 which storeselectronic data concerning, for example, academic records, homeenvironment, health conditions, or the age of each pupil or student. Thepupil or student information server computer 72 is connected to theterminal computer 71 via a LAN, and is configured such that internalbiological time information downloaded from the server computer 11 tothe terminal computer 71 can be input to the pupil or studentinformation server computer 72.

The pupil or student information server computer 72 may be configuredsuch that internal biological time information pertaining to each pupilor student can be automatically and directly incorporated to informationpertaining to each pupil or student. This can be realized by allottingto each pupil or student, as his/her test subject number (describedhereinbelow), his/her pupil or student ID number registered in the pupilor student information server computer 72, and by installing a programwhich automatically transfers, to an unillustrated database provided inthe pupil or student information server computer 72, the internalbiological time information pertaining to each pupil or studentdownloaded from the server computer 11 to the terminal computer 71.

The educational institution 7 may be replaced by a research organizationsuch as a university or a laboratory.

The clients 2 to 7 collect blood samples from test subjects and placethe samples in test tubes provided by the information center 1. Thesamples will hereafter be referred to as blood samples 19. At the timewhen the blood samples are placed in the test tubes, each test tube islabeled with a client ID number given uniquely to each client, a testsubject number identifying the test subject, and year/month/day and timeat which the sample was drawn. The clients 2 to 7 submit the bloodsamples 19 to the information center 1 by mail or walk-in.

One or two days after submission of the blood samples 19, the clients 2to 7 can view, on terminal computers 21 to 71, the judgment results asto whether or not the individual test subjects suffer circadian rhythmdisorders and, in the case of the absence of circadian rhythm disorders,internal biological times of respective test subjects.

When the clients 2 to 7 each have a gene product quantity measuringapparatus, the clients 2 to 7 can measure on their side mRNA expressionlevels of the blood samples 19 and transfer, from the terminal computers21 to 71 to the server computer 11, the relevant measurement datatogether with information including the corresponding client ID numberswhich have been given in advance, the corresponding test subjectnumbers, and the year/month/day and time at which the samples weredrawn.

Within several hours from transfer of the measurement data, the clients2 to 7 can view, on terminal computers 21 to 71, the judgment results asto whether or not the individual test subjects suffer circadian rhythmdisorders and, in the case of the absence of circadian rhythm disorders,internal biological times of respective test subjects.

The apparatuses and computer will next be described.

A DNA chip reader 12 may be a known DNA chip reader and includes a DNAchip scanner (not shown), a CPU (not shown) for processing the data readby the scanner, and a storage for storing the processed data.

The DNA chip reader 12 measures the amounts of respective mRNA speciesamong the total RNA contained in a specimen, and the measurement dataare stored in the storage (not shown).

Although the present embodiment employs a DNA chip reader 12 to measurethe quantity of a gene product, there may be employed any other suitableapparatuses for, for example, quantitative PCR, real-time PCR, DNAmicroarray assays, RNase protection assays, or northern hybridizationassays.

When a protein level measurement is desired to be used as the quantityof a gene product, there may be employed a known apparatus capable ofperforming, for example, two-dimensional electrophoresis, massspectroscopy, protein chip analysis, antibody chip analysis, orimmunoblotting.

The server computer 11 determines, by use of the gene product quantitydata, whether or not the specimen indicates circadian rhythm disordersand estimates the internal biological time of the test subject relatedto the specimen.

The server computer 11, as shown in FIG. 2, is equipped with a centralprocessing unit (CPU 70), storage devices (RAM 73, ROM 74, HDD 75, and astorage medium device 76), a communication device 77, a keyboard 78, amouse 79, a display 80, and a printer 81.

The CPU 70 performs calculations by use of the information from thestorage devices, the communication device, and the input device, andplays a role to transmit the calculation results to the storage devices,the communication device, and the output device.

The storage devices store programs and the like to perform variousprocesses. Among the storage devices, the storage medium device 76includes external medium devices such as HDD, CD, and DVD, and, asoccasion requires, can store or read out data received via thecommunication device 77 or data input through the keyboard 78 or themouse 79.

The RAM 73 stores data, etc. which are necessary for the CPU 70 toperform processes. The display 80 displays the image or the like whichthe CPU 70 created with programs stored in the ROM 74, HDD 75, or thelike. The printer 81 outputs predetermined items of information fromrespective computers on paper. The communication device 77transmits/receives data to and from other computers including the DNAchip reader 12 and the terminal computers 21 to 71.

The HDD 75 contains the following items: a time-course gene productquantity table 14 as shown in FIG. 3; a circadian oscillatory gene table(not shown) including circadian oscillatory gene candidates' time-coursegene product quantity data chosen from standard specimen's time-coursegene product quantity data; a molecular timetable 15 as shown in FIG. 4,an internal biological time information table 16 as shown in FIG. 5;cosine wave information for selecting genes (not shown), and cosine waveinformation for generating timetables (not shown).

The time-course gene product quantity table 14, as shown in FIG. 3, is atable where the time-course data regarding the quantity of a geneproduct contained in a standard specimen is registered for each gene.

The molecular timetable 15, as shown in FIG. 4, is a table to be used todetermine whether or not the sample shows the presence of a circadianrhythm disorder and to estimate internal biological time through theestimating process. The molecular timetable is formed of numerical valueitems that characterize the circadian expression curve of each of thecircadian oscillatory genes chosen from a standard specimen; i.e.,standard expression level 153, standard variation 154, and moleculartime 155, all these three items being determined under LD conditions(24-hour cyclic light/dark conditions with a 12-hour light period and a12-hour dark period), as well as standard expression level 156, standardvariation 157, and molecular time 158, all these three items beingdetermined under DD conditions (constant dark conditions).

The internal biological time information table 16, as shown in FIG. 5,is a table where the judgment concerning whether or not the sample froma test subject shows a circadian rhythm disorder and the results fromthe estimation process for determining the internal biological time ofthe sample from the test subject are tabulated as records. Therespective records are registered when the process for estimatinginternal biological time and providing information, as shown in theflowchart of FIG. 10, is executed.

The internal biological time information table 16 is formed of thefollowing registration items: entry number 161 allotted to each specimenby the information center 1 upon receipt of a blood sample 19, clientnumber 162 allotted to each client at the time of the client'smembership registration, user ID 163 of a person in charge at theclient, user password 164 of the person in charge, test subject number165 allotted to each test subject on the client's side, samplecollection time 166 which is indicated by way of labeling on the testtube of the blood sample 19 at the time of collection of the bloodsample 19 on the client's side and which is input to a computer at theinformation center 1, judgment as to the presence or absence ofcircadian rhythm disorders 167 rendered by the server computer 11,internal biological time estimation results 168 rendered by the servercomputer 11, and comments 169 entered as needed in relation to eachspecimen on the information center 1's side.

The cosine wave information for selecting genes is information in theform of cosine waves which are used for selecting circadian oscillatorygenes selected from among the genes contained in a standard specimen.

The number of the cosine waves for gene selection is 540; i.e., thereare 540 cosine waves having different periods of 20 to 28 hours withincrements of 1 hour and with phases shifted by one sixtieth the period.That is, for each of the 9 periods, 60 cosine waves are drawn with theirphases shifted. A portion of these cosine waves to be used for geneselection are shown in FIG. 8.

The cosine wave information for gene selection comprises the followingformula A(t) used for calculating a value z(t) on a cosine wave for geneselection: $\begin{matrix}{{A(t)} = {{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{Period} \right)}} & (6)\end{matrix}$where

-   period: 9 different hours; i.e., 20, 21, 22, 23, 24, 25, 26, 27, and    28-   T: each point in time where a period is divided into 60 equal    portions and where the cosine wave reaches the maximum value-   t: a certain point in time

The cosine wave information for generating timetables is information inthe form of cosine waves which are used for generating moleculartimetables of circadian oscillatory genes selected from among the genescontained in a standard specimen.

The number of the cosine waves for timetable generation is 144; i.e.,there are 144 cosine waves (period of each wave: 24 hours) having phasesshifted by 10 minutes.

The cosine wave information for timetable generation comprises thefollowing formula A(t) used for calculating a value α(t) on a cosinewave for timetable generation: $\begin{matrix}{{A(t)} = {{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{Period} \right)}} & (7)\end{matrix}$where

-   period: 24 (hours)-   T: molecular time on the cosine wave (for example, 144 different    points in time with intervals of 10 minutes (0/60, 10/60, 20/60,    30/60, . . . , 1420/60, and 1430/60))-   t: a certain point in time

Next, based on FIG. 6, the process flow of the internal biological timeestimating system of the present embodiment will be described.

Information center 1 provides, at the server computer 11, internalbiological time information access services for clients via Internet 13.

As shown in FIG. 6, the process performed by the internal biologicaltime estimating system generally includes a gene selection/timetablegeneration stage 100, a circadian rhythm disorder judgment/internalbiological time estimation stage 200, and a reporting-to-clients stage300.

The gene selection/timetable generation stage 100 is a preparatory stagefor the circadian rhythm disorder judgment/internal biological timeestimation stage, and is performed before an internal biological timeinformation provision service is available to a client.

The portion—or in other words—type of the tissue/cells used in circadianrhythm disorder judgment/internal biological time estimation and acircadian oscillatory gene are selected, and then a molecular timetableof the circadian oscillatory gene is generated.

In the gene selection/timetable generation stage 100, firstly,time-course gene expression data of standard specimens are obtained instep 101. This data acquisition is carried out by use of a DNA chipreader 12.

The standard specimens are biological tissues sampled from a pluralityof biological individuals. In the present embodiment, the standardspecimens are blood drawn from a plurality of humans.

As the standard specimens, blood is sampled at predetermined timeintervals from a plurality of humans under predetermined conditions.Specifically, after a plurality of examinees are forced to stay in aroom under a 24-hour cycle of 12-hour-light and 12-hour-dark conditionsfor 2 weeks, blood is drawn every 4 hours over 2 days under the same24-hour cycle of 12-hour-light and 12-hour-dark conditions (LDconditions) or under constant dark conditions (DD conditions).

Blood sampling under LD conditions is performed at the following 12points in time: immediately before lights on (ZT0), 4 hours after lightson (ZT4), 8 hours after lights on (ZT8), immediately before lights off(ZT12), 4 hours after lights off (ZT16), 8 hours after lights off(ZT20), and on Day 2, immediately before lights on (ZT24), on Day 2, 4hours after lights on (ZT28), on Day 2, 8 hours after lights on (ZT32),on Day 2, immediately before lights off (ZT36), on Day 2, 4 hours afterlights off (ZT40), and on Day 2, 8 hours after lights off (ZT44). Bloodsampling under DD conditions are the following 12 points in time:immediately before the start of DD conditions (CT0), 4 hours after thestart of subjective daytime (CT4), 8 hours after the start of subjectivedaytime (CT8), immediately before the start of subjective night (CT12),4 hours after the start of subjective night (CT16), 8 hours after thestart of subjective night (CT20), and on Day 2, immediately before thestart of subjective daytime (CT24), on Day 2, 4 hours after the start ofsubjective daytime (CT28), on Day 2, 8 hours after the start ofsubjective daytime (CT32), on Day 2, immediately before the start ofsubjective night (CT36), on Day 2, 4 hours after the start of subjectivenight (CT40), and on Day 2, 8 hours after start of subjective night(CT44).

Blood samples drawn from a plurality of humans are treated with knownmethods to thereby prepare, for each individual, each of the LD/DDconditions, and each sampling time, a standard specimen for measuringthe mRNA quantity, which is a gene product quantity.

The mRNA quantity of a standard specimen—prepared for each individual,each of the LD/DD conditions, and each sampling time—is measured by useof a DNA chip reader 12, whereby time-course gene product quantity dataof respective standard specimens are obtained.

The obtained time-course gene product quantity data are stored within astorage device (not shown) of the DNA chip reader 12, and transmittedtherefrom to a server computer 11.

Next, in step 102, on the basis of the time-course gene product quantitydata received in step 101, the server computer 11 selects circadianoscillatory genes.

The procedure for selecting circadian oscillatory genes is described indetail with reference to the circadian oscillatory gene selectionflowchart shown in FIG. 7. The procedure of FIG. 7 is controlled by aCPU 70 of the server computer 11.

The server computer 11 receives time-course gene product quantity datafrom the DNA chip reader 12, and stores the data as a time-course geneproduct quantity table 14 on HDD 75. FIG. 3 shows the time-course geneproduct quantity table 14.

In the time-course gene product quantity table 14, the mRNA quantitywhich is associated with the expression level of a gene is registered ateach point in time for each gene.

When the time-course gene product quantity table 14 is registered on HDD75, the process starts following the flowchart shown in FIG. 7. Firstly,in step S101, one gene record item is read out from the time-course geneproduct quantity table 14.

Subsequently, in Step S102, one value for period and one value for T aresubstituted into the following formula z(t) which is derived by applyingthe standard variation and standard expression level of each gene toformula A(t) of the cosine wave information for gene selection acquiredfrom HDD 75, to thereby specify one cosine wave from among 540 cosinewaves. $\begin{matrix}{{z(t)} = {{\frac{A}{B}{{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{Period} \right)}} + C}} & (8)\end{matrix}$where

-   Period: 9 different hours; i.e., 20, 21, 22, 23, 24, 25, 26, 27, and    28-   A: standard variation of gene A-   B: standard deviation of cosine wave calculated from formulas (4)    and (5) given hereinabove-   C: standard expression level of gene A-   T: when the period is divided into 60 equal portions, the point in    time at which the cosine wave shows the maximum value,-   t: a certain point in time.

Next, in step S103, a Pearson's product moment correlation coefficientis calculated between each of the values as determined at respectivepoints in time of the gene record read in step S101 and each valueobtained by substituting a corresponding 4-hour-interval point in timefor t of formula z(t), and the calculation results are stored in RAM 73.

That is, a Pearson's product moment correlation coefficient iscalculated between the value xn at time 4n in the gene record (i.e., theexpression level at ZT4n or the expression level at CT4n, where n is aninteger of 0 or more and 11 or less) and the value yn on a cosine waveat time 4n (i.e., the value on the cosine wave at ZT4n or the value onthe cosine wave at CT4n, where n is an integer 0 or more and 11 or less)is calculated.

Next, in step S104, judgment is made as to whether or not there remainsany period or T for which product moment correlation coefficient has notbeen calculated.

In the case where there remain periods and T for which product momentcorrelation coefficient has not been calculated (step S104: YES), instep S102, one value for period and one value for T are substituted intoformula z(t) to thereby specify one cosine wave from among 540 cosinewaves.

If there remains no period or T for which product moment correlationcoefficient has not been calculated (step S104: NO), calculation andstorage of product moment correlation coefficients are considered tohave been completed for all the cosine waves for selection of genes, andin step S105, a determination is made as to whether or not there is anycoefficient that is greater than 0.75 among all the product momentcorrelation coefficients that were calculated.

If there is found a coefficient value that is greater than 0.75 (stepS105: YES), since the relevant gene is regarded as having an expressionpattern oscillating with a period of between 20 to 28 hours inclusiveand thus exhibiting a characteristic of the circadian oscillatory gene,in step S106, a gene candidate flag is set on its gene record.

Next, step S107 decides on whether or not there remains any gene recordthat has not yet undergone the process for calculating product momentcorrelation coefficient with cosine wave for gene selection. The productmoment correlation coefficient calculation process is performed for eachgene for both gene records; i.e., gene records under LD conditions andDD conditions.

If there remains a gene record that has not yet undergone thecorrelation coefficient calculation process (step S107: YES), in stepS101, one gene record is read.

In the case where there remains no gene record that has not undergonethe correlation coefficient calculation process (step S107: NO), all thegenes are regarded as having undergone the judgment process fordetermining whether or not they are circadian oscillatory genes, and instep S108, an unillustrated circadian oscillatory gene table havingcircadian oscillatory gene candidates is generated. In step S108,referring to the time-course gene product quantity table 14, only suchgene records that have gene candidate flags for both LD and DDconditions are extracted, to thereby generate an unillustrated circadianoscillatory gene table. The process is then terminated.

With the process described above, selection for circadian oscillatorygenes in process 102 in FIG. 6 is completed.

Upon completion of the circadian oscillatory gene selection in process102 in FIG. 6, in process 103, the server computer 11 generates acircadian expression curve and a molecular timetable for each geneproduct.

This process is described with reference to the circadian expressioncurve and molecular timetable generation flowchart shown in FIG. 9. Theprocess of FIG. 9 is controlled by the CPU 70 of the server computer 11.

Once the selection of circadian oscillatory genes is completed inprocess 102 in FIG. 6, the flowchart of FIG. 9 starts.

Firstly, in step S201, for each of the LD and DD conditions, onecircadian oscillatory gene record is read out from the unillustratedcircadian oscillatory gene table produced in step S108 in FIG. 7.

Next, in step S202, one value for T is substituted into the followingformula α(t) which is derived by applying the standard variation andstandard expression level of each gene to formula A(t) of the cosinewave information for timetable generation acquired from HDD 75, tothereby specify one cosine wave from among 144 cosine waves.$\begin{matrix}{{\alpha(t)} = {{\frac{A}{B}{{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{24} \right)}} + C}} & (9)\end{matrix}$

-   A: standard variation of circadian oscillatory gene A-   B: standard derivation of cosine wave calculated from formulas (4)    and (5) given hereinabove-   C: standard expression level of gene A-   T: molecular time on cosine wave (for example, the time T may be    chosen from 144 points in time; i.e., 0/60, 10/60, 20/60, 30/60, . .    . , 1420/60, and 1430/60, in increments of 10 minutes), and-   t: a certain point in time.

Next, in step S203, a Pearson's product moment correlation coefficientis calculated between each of the values as determined at respectivepoints in time of the circadian oscillatory gene record read in stepS201 and each value obtained by substituting a corresponding4-hour-interval point in time for t of formula α(t), and the calculationresults are stored in RAM 73.

That is, a Pearson's product moment correlation coefficient iscalculated between the value xn at time 4n in the circadian oscillatorygene record (i.e., the expression level at ZT4n or the expression levelat CT4n, where n is an integer of 0 or more and 11 or less) and thevalue yn on a cosine wave at time 4n (i.e., the value on the cosine waveat ZT4n or the value on the cosine wave at CT4n, where n is an integer 0or more and 11 or less) is calculated.

Next, in step S204, judgment is made as to whether or not there remainsany T for which product moment correlation coefficient has not beencalculated.

In the case where there remain T for which product moment correlationcoefficient has not been calculated (step S204: YES), in step S202, oneT value is substituted into formula z(t) to thereby specify one cosinewave from among 144 cosine waves.

If there remains no T for which product moment correlation coefficienthas not been calculated (step S204: NO), calculation and storage ofproduct moment correlation coefficients are considered to have beencompleted for all the cosine waves for timetable generation with regardto the circadian oscillatory gene record under the relevant LD/DDconditions, and in step S205, the cosine wave which provides the maximumvalue among all the product moment correlation coefficients calculatedfor the circadian oscillatory gene record under the relevant LD/DDconditions is extracted and stored in RAM 73. This cosine wave is acircadian expression curve exhibiting an oscillatory expression patternof the circadian oscillatory gene.

Subsequently, in step S206, the point in time at which the value on thecircadian expression curve, which is extracted from the cosine waves,exhibits the maximum is registered in the molecular timetable 15 shownin FIG. 4 along with the name of the circadian oscillatory gene, theconditions (LD or DD), the standard expression levels, and the standardvariations.

The standard expression levels 153 and 156, the standard variations 154and 157, and the molecular times 155 and 158 characterize the circadianexpression curve.

Subsequently, in step S207, a determination is made as to whether or notthere remain any items of a circadian oscillatory gene record which hasnot undergone processing for calculation of a product moment correlationcoefficient with the cosine wave for generating a timetable.

If there remain any items of a circadian oscillatory gene record whichhas not undergone processing for calculation of a product momentcorrelation coefficient with the cosine wave for generating a timetable(step S207: YES), in step S201, one circadian oscillatory gene record isread, for each of LD and DD conditions, from a circadian oscillatorygene table (not shown) generated in step S108 in FIG. 7.

If there remain no items of a circadian oscillatory gene record whichhas not undergone processing for calculation of a product momentcorrelation coefficient with the cosine wave for generating a timetable(step S207: NO), procedures for generating circadian expression curvesand molecular timetables are considered to have been completed for allthe circadian oscillatory genes, and the process is terminated.

Through the above steps, the process 103 in FIG. 6 for generatingcircadian expression curves and molecular timetables for respectivegenes is completed.

Thus, the gene-selection/timetable-generation stage 100 is completed,and services for providing internal biological time information toclients are now available.

Upon completion of the gene-selection/timetable-generation stage 100,the information center 1 starts services for providing internalbiological time information.

Users at clients 2 to 7 who are desirous of using the internalbiological time estimating system of the present embodiment obtain amembership status with the information center 1 in advance.

Membership registration is done by inputting, on a membershipregistration screen provided by the server computer 11, information suchas member organization name, user name, user password, e-mail address ofthe user, address for contact, and manner of payment for service fees.

In the present embodiment, the internal biological time informationproviding service is a service for members registered for membership inadvance. However, services may be provided by selling a kit including atest tube for a blood sample 19 and an instruction manual for connectingto the server computer 11 in an information center 1 so as to allow aperson who purchases the kit to have an one-time service.

The clients 2 to 7 draw blood from patients, place in test tubesprovided by the information center 1, and send blood samples 19 to theinformation center 1. The information center 1 measures mRNA expressionlevels of the blood samples 19 with DNA chip readers 12 through a knownmethod.

Upon completion of the expression level measurement of a blood sample19, the information center 1 activates the server computer 11 to therebystart processing for circadian rhythm disorder determination, internalbiological time estimation, and information provision.

The processing performed in the server computer 11 for determining onthe presence or absence of circadian rhythm disorders, estimatinginternal biological time, and providing information will be describedwith reference to the flowcharts shown in FIGS. 10 to 13.

When the process is started, an initial setting is made in step S301.The initial setting process of step S301 will next be described indetail referring to the flowchart in FIG. 11.

In the initial setting process in step S301, firstly, in step S351, athreshold value D that has been input by a user of the server computer11 through a keyboard 78 or the like is input and stored in anunillustrated threshold value memory area in the HDD 75.

The threshold value D is a decisive value for determining the presenceof circadian rhythm disorders; if the maximum value C of the calculatedPearson's product moment of correlation coefficients between wavesrepresenting the expression levels of circadian oscillatory geneproducts in a sample and waves representing the estimated expressionlevels of the circadian oscillatory gene products is below the thresholdvalue D, the presence of circadian rhythm disorders is determined.

Preferably, the threshold value D differs depending on the number ofcircadian oscillatory genes in the sample. For example, as thresholdvalue D, a smaller value may be employed for a higher number ofcircadian oscillatory gene species contained in a sample. When thenumber of the circadian oscillatory gene species in the sample is about100 to 200, a threshold value D of 0.3 or more, preferably 0.5 or moreis employed.

In the present embodiment, the threshold value D is 0.5.

Subsequently, in step S352, a threshold value U entered through thekeyboard 78 or the like by a user of the server computer 11 is input andstored in an unillustrated threshold value memory area in the HDD 75.

The threshold value U is a decisive value for determining the presenceof circadian rhythm disorders; the value represents the time shiftbetween the internal biological time as estimated for a circadianoscillatory gene in a sample and the environmental time. In the presentembodiment, the threshold value U is 2 hours.

Next, in step S353, standard expression levels, standard variations, andthe molecular times are input for each of the gene products of thestandard specimens. In this step, molecular timetables corresponding tothe type of the sample to be measured are selected from among themolecular timetables 15 generated through thecircadian-expression-curve/molecular-timetable generation process(process 103 of FIG. 6) for each gene product, and stored in ROM 74.

Subsequently, in step S354, values of standard expression levels,standard variations, and molecular times are extracted from themolecular timetables stored in step S353 corresponding to the type ofthe sample to be measured, and the circadian expression curves of thestandard specimens represented by the following equation are derived.$\begin{matrix}{{\alpha(t)} = {{\frac{A}{B}{{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{24} \right)}} + C}} & (10)\end{matrix}$where

-   A: standard variation of a circadian oscillatory gene A,-   B: standard deviation of the cosine wave calculated from    equations (4) and (5),-   C: standard expression level of gene A,-   T: molecular time of the cosine wave (for example, the following 144    different points in time in increments of 10 minutes; i.e., 0/60,    10/60, 20/60, 30/60, . . . , 1420/60, and 1430/60), and-   t: a certain point in time.

Subsequently, in step S355, the circadian expression curves arenormalized. Specifically, a relative circadian expression curverepresented by the following equation is derived by subtracting astandard expression level from a relevant circadian expression curve foreach of the circadian oscillatory genes of the standard specimens,followed by division by standard variation. $\begin{matrix}{{\beta(t)} = {\frac{1}{B}{{Cos}\left( \frac{2{\pi\left( {t - T} \right)}}{24} \right)}}} & (11)\end{matrix}$where

-   B: standard deviation of the cosine wave calculated from    equations (4) and (5),-   T: molecular time of the cosine wave (for example, 144 different    points in time in increments of 10 minutes; i.e., 0/60, 10/60,    20/60, 30/60, . . . , 1420/60, and 1430/60), and-   t: a certain point in time.    The thus-obtained relative circadian expression curves are stored in    ROM 74.

Through the above steps, the initial setting procedure in step S301 inFIG. 10 is terminated.

Subsequently, in step S302 in FIG. 10, the sampling time S of the sampleof interest entered by a user on the server computer 11 is stored in RAM73. The sampling time is an environmental time.

Next, in step S303, the circadian oscillatory gene product quantity dataof the sample is registered in HDD 75. This step is performed byreceiving the circadian oscillatory gene product quantity data of thesample from the DNA chip reader 12 operated by the user on the servercomputer 11.

Subsequently, in step S304, normalization of the circadian oscillatorygene product quantity data of the sample is performed, and relativeexpression levels for each of the circadian oscillatory gene productsare determined. The process in step S304 will next be described indetail with reference to FIG. 12.

In step S361, the standard expression level of the standard specimens issubtracted from the circadian oscillatory gene product quantity data ofthe sample. The values of the standard expression level have beenobtained through calculation of average values of circadian oscillatorygene product quantity data of the standard specimens for each of thegenes.

In step S362, the values obtained in step S361 are divided by therelevant standard variation of the standard specimens. The values of thestandard variation have been obtained through calculation of thestandard deviation of circadian oscillatory gene product quantity dataof the standard specimens for each of the genes.

Through the above steps, the normalization process in step S304 for thecircadian oscillatory gene product quantity data is completed, andrelative expression levels for each of the circadian oscillatory geneproducts of the sample are obtained.

Next, in step S305, a value 0 is substituted into t in equation β(t)representing the relative circadian expression curves of the standardspecimens, and values on the relative circadian expression curves foreach of the circadian oscillatory genes at t=0 are computed.

Subsequently, in step S306, a determination is made as to whether or notthe value substituted into t in equation β(t) representing the relativecircadian expression curves of the standard specimens is larger than 24.

If the value substituted into t in equation β(t) representing therelative circadian expression curves of the standard specimens is notlarger than 24 (step S306: NO), or in other words, if the valuesubstituted into t in equation β(t) representing the relative circadianexpression curves of the standard specimens falls within a range of 0 to24 inclusive, in step S307, Pearson's correlation coefficient c betweena normalized circadian oscillatory gene product quantity and anestimated relative expression level of each of the circadian oscillatorygene products at time t is computed.

The process in step S307 will next be described in detail with referenceto FIG. 13.

Firstly, in step S371, for each of the circadian oscillatory genes, anestimated relative expression level at time t on the relative circadianexpression curve of the gene is obtained. In this process, the moleculartimetable records for the respective circadian oscillatory genes areread one by one, and relative circadian expression curves for thecircadian oscillatory genes are obtained. Subsequently, a value on arelative circadian expression curve is obtained at time t and employedas the estimated relative expression level. This process is performedfor each and every circadian gene.

Subsequently, in step S372, the Pearson's product moment correlationcoefficient between the relative expression levels of circadianoscillatory gene products of the sample at the sampling time which hasbeen normalized in step S304 and the estimated relative expressionlevels at time t on the relative circadian expression curves for thecircadian oscillatory genes is computed. Each computed value is storedin RAM 73 together with the value of time t, and the process shown inFIG. 13 is completed.

Subsequently, the process proceeds to step S308 in FIG. 10, and dt isadded to the previous time t. The value t+dt is substituted into t inequation β(t) which represents the relative circadian expression curvesof the standard specimens, and the values on the relative circadianexpression curves for each of the circadian oscillatory genes at t=t+dtare computed.

In the present embodiment, a value of 10 minutes or 10/60 hours isemployed for dt.

Subsequently, the process proceeds to step S306, and a determination ismade as to whether or not the value substituted into t in equation β(t)representing the relative circadian expression curves of the standardspecimens is larger than 24.

If the value substituted into t in equation β(t) expressing the relativecircadian expression curves of the standard specimens is larger than 24(step S306: YES), calculation of Pearson's product moment correlationcoefficient is considered to have been completed between the relativeexpression levels for the circadian oscillatory gene products of thesample and the estimated relative expression levels at every point intime between 0 and 24 on the relative circadian expression curves forthe circadian oscillatory genes, and thus, the process proceeds to stepS309, whereby the maximum value C of the Pearson's product momentcorrelation coefficients c and the internal biological time T whichgives the maximum value C are retrieved.

In this step, a set of data for the Pearson's product moment correlationcoefficients c stored in RAM 73 is read, and the maximum Pearson'sproduct moment correlation coefficient value is extracted from the dataset and employed as the maximum value C. Subsequently, the timecorresponding to the maximum value C of the Pearson's product momentcorrelation coefficient is read and employed as the internal biologicaltime T that gives the maximum value C. The maximum value C and theinternal biological time T are registered in RAM 73 as a maximum value Cand an internal biological time T of the sample.

Subsequently, in step S310, a determination is made as to whether themaximum value C stored in RAM 73 satisfies the following condition“C<threshold value D.” Since a threshold value D of 0.5 has beenregistered in the present embodiment, in this step, a determination ismade as to whether or not the maximum value C is smaller than 0.5.

If the maximum value C is smaller than the threshold value D (step S310:YES), then the relative expression levels of circadian oscillatory geneproducts of the sample at a certain time at which the sample was drawndo not exhibit similarity with any of the estimated relative expressionlevels calculated for the circadian oscillatory genes estimated atvarious times. Thus, the test subject from whom sample has been drawn isconsidered to have circadian oscillatory genes which do not exhibitnormal circadian oscillations. In step S311, circadian rhythm disorderis diagnosed on this sample, and a value “1,” which indicates thepresence of rhythm disorders, is registered in circadian rhythm disorderpresence/absence determination registry 167 of the corresponding entrynumber in the internal biological time information table 16.

If the condition “maximum value C<threshold value D” is not met (stepS310: NO), or in other words, if the maximum value C is equal to orlarger than the threshold value D, then there exists a time T at whichthe relative expression levels of circadian oscillatory gene products ofthe sample at a certain time at which the sample was drawn exhibitssimilarity with estimated relative expression levels estimated for thecircadian oscillatory genes. Thus, the test subject from whom thissample has been drawn possibly has circadian oscillatory genesexhibiting normal circadian oscillations, and therefore, the processproceeds to step S312, and a determination is made as to whether theabsolute value of the difference between internal biological time T andsampling time S satisfies the following relation.|T−S|>threshold value U  (12)Since a threshold value U of 2 hours has been registered, adetermination is made as to whether the absolute value of the differencebetween the internal biological time T and the sampling time S is largerthan 2 hours.

If the absolute value of the difference between the internal biologicaltime T and the sampling time S is larger than the threshold value U(step S312: YES), the time shift from the sampling time S, which is anenvironmental time, is considered to be too large even though generalerrors which may be involved in the internal biological time estimationmethod of present invention are taken into account. Thus, in step S313,the presence of circadian rhythm disorders is diagnosed on this sample,and a value “1,” which indicates rhythm disorders, is registered incircadian rhythm presence/absence determination registry 167 of thecorresponding entry number in the internal biological time informationtable 16.

Subsequently, in step S314, the internal biological time T calculated instep S309 is stored in internal biological time estimation registry 168under the corresponding entry number in the internal biological timeinformation table 16, and the process is terminated.

If the absolute value of the difference between the internal biologicaltime T and the sampling time S is not larger than the threshold value U(step S312: NO), or in other words, if the absolute value of thedifference between the internal biological time T and the sampling timeS is equal to or smaller than the threshold value U, the time shift fromthe sampling time S, which is an environmental time, is considered tofall within a normal range. In this case, value T is considered to be areasonable internal biological time of the test subject, and theinternal biological time T is registered in the internal biological timeestimation registry 168 under the corresponding entry number in theinternal biological time information table 16, and the process isterminated.

Through the above steps, the circadian rhythm disorder determinationprocess and the internal biological estimation process are terminated.

A process for permitting clients 2 to 7 to have an access, by way ofviewing, to information as to the presence or absence of circadianrhythm disorders or internal biological time information, which arestored in the server computer 11, will next be described.

Upon completion of thecircadian-rhythm-disorder-determination/internal-biological-time-estimationstage 200 shown in FIG. 6, users of clients 2 to 7 who have sent theblood samples 19 of test subjects to the information center 1 receivefrom the server computer 11 e-mail notifying completion of the circadianrhythm disorder determination process and the internal biologicalestimation process.

After a user is advised of completion of the circadian rhythm disorderdetermination process and the internal biological estimation processthrough e-mail, the user receives a report 300, shown in FIG. 6, ofinternal biological time information on an internal biological timeinformation report screen 91 provided by the server computer 11.

The process of FIG. 6 for reporting internal biological time informationon the internal biological time information report screen 91 will nextbe described.

When the address of an internal biological time information providingservice screen of the information center 1 is input via the Internet atthe terminal computer 21, an unillustrated initial screen of theinternal biological time information providing service is displayed.

In the initial screen, information on internal biological time and othergeneral information items regarding drug dosage time management, etc.are displayed, as well as an ID/password input screen display button forthe user to move to a “members only” screen for access to the internalbiological time information providing service.

When the ID/password input screen display button is clicked, anID/password input screen (not shown) is displayed. On this screen, whenan ID and a password are input, the server computer 11 searches theinternal biological information table 16 using the client ID number 162for the client as a key item, extracts the records of the client, andgenerates data to be shown on the internal biological time informationreport screen 91.

Thereafter, the data to be shown on the internal biological timeinformation report screen 91 is transmitted to the terminal computer 21.

When the user enters his or her membership ID on the “members only”screen provided by the terminal computer 21 for log-in, the internalbiological time information report screen 91 as shown in FIG. 14 isdisplayed.

On the internal biological time information report screen 91, thefollowing are displayed: an internal biological time information list910 for the specimen on which the log-in member requested adetermination on the presence or absence of rhythm disorders and aninternal biological time estimation, a close button (or click box) 921for closing the internal biological time information report screen 91, adownload button 922 for downloading the determination or estimationresults for a specimen check-marked in a download check box 919, and alog-out button 923.

The internal biological time information list 910 is a list prepared foreach specimen and containing internal biological information of thespecimen, and the following items are displayed for each specimen: entrynumber 911 of the specimen, test subject number 912 from whom thespecimen has been drawn, sampling time 913 at which the specimen wasdrawn from the test subject, reception date 914 indicating the date onwhich the information center 1 received the specimen, the presence orabsence of rhythm disorders 915 as a test result of the specimen,internal biological time information 916 as a test result of thespecimen, a history screen display button 917 for the history regardinginternal biological time information under the test subject number, acomment display button 918 for the test results of the specimen, and adownload check box 919 for downloading the test results of the specimen.

By clicking the history screen display button 917, a user can view, inthe list displayed, the history of the results as determined in the paston a test subject from whom the specimen has been drawn.

The server computer 11 may store a program for performing a dataanalysis on a large number of test subjects' internal biological timeinformation data provided by the internal biological time estimationsystem.

This program may be configured so as to enable analysis regarding thecorrelation between internal biological time of a human subject andcharacteristics of that human subject through derivation of categorizedinternal biological time information by age, sex, ethnicity,pathological conditions, physical characteristics such as height andbody weight, type of work, etc. The analysis results of correlationbetween human internal biological time and human characteristics may beutilized for human beings' health promotion and measures againstdiseases.

Next will be described the processing for data analysis on internalbiological time information of a large number of test subjects obtainedthrough provision of the internal biological time estimation system.

When the information center 1 receives a blood sample 19 along with atest subject number 165, the center 1 also receives characteristic datasuch as the date of birth, sex, ethnicity, pathological conditions, andheight and body weight of the test subject.

These data may be received via the Internet 13 at the time of receipt ofthe blood sample 19. Alternatively, the blood sample 19 may beaccompanied by a sheet of paper on which the data are shown.

The respective test subjects' characteristic data received by theinformation center 1 are stored in a test subject characteristic table17 at the server computer 11, as shown in FIG. 15.

If the number of record items stored in the internal biological timeinformation table 16 and the test subject characteristic table 17 hasreached a predetermined number, the server computer 11 data starts dataanalysis.

Example data analysis results are shown in FIG. 16.

The results of data analysis may be used later for diagnosis of thesleep-wake rhythm disorders (delayed sleep phase syndrome, advancedsleep phase syndrome, non-24 hour circadian rhythm sleep-wake disorder,etc.), seasonal depression, and jet-lag syndrome.

No particular limitation is imposed on the type of the circadianexpression curve, insofar as it can be expressed by a mathematicalformula of a time-course expression profile of a circadian oscillatorygene derived on the basis of a finite number of expression data, orexpression level data, which have been obtained over a certain period oftime.

For example, the circadian expression curve may be generated through amethod for creating a periodic curve, utilizing the Fourier transform,of time-course expression level data of each of the circadianoscillatory genes (Reference 1). The “Fourier transform” is a transformbased on the principle first formulated by Frenchmathematician-physicist Fourier, and is used for bridging twomathematical expressions (note: a physical process can be expressedeither as a function of time; i.e., in the form of h(t), or as afunction of frequency f; i.e., in the form of H(f)). The Fouriertransform is represented by the following expressions: $\begin{matrix}{{h(t)} = {\int_{- \infty}^{\infty}{{H(f)}{\exp\left( {{- 2}\pi\quad{\mathbb{i}}\quad f\quad t} \right)}\quad{\mathbb{d}f}}}} & (13) \\{{H(f)} = {\int_{- \infty}^{\infty}{{h(t)}{\exp\left( {2\pi\quad{\mathbb{i}f}\quad t} \right)}\quad{\mathbb{d}t}}}} & (14)\end{matrix}$

The Fourier transform for a finite number of sample values(measurements) is called discrete Fourier transform. Suppose that thereare a series of measurements in a number N which are to undergo adiscrete Fourier transform:h _(k) ≡h(t _(k)), t _(k) ≡kΔ, k=0, 1, 2, . . . , N−1The sample interval is Δ. Here, the discrete Fourier transform isdefined as follows: $\begin{matrix}{H_{n} \equiv {\sum\limits_{k = 0}^{N - 1}\quad{h_{k}\quad{\exp\left( \frac{2\pi\quad{\mathbb{i}k}\quad n}{N} \right)}}}} & (15)\end{matrix}$where

-   n=0, 1, 2, . . . , N−1

By use of Hn, the following function of time, h(t), can be estimated:$\begin{matrix}{{h(t)} \approx {\frac{1}{N}{\sum\limits_{n = 0}^{N - 1}\quad{H_{n}\quad{\exp\left( {{- 2}\pi\quad{\mathbb{i}}\frac{n}{N\quad\Delta}t} \right)}}}}} & (16)\end{matrix}$

A circadian expression curve may alternatively be generated according toa method for creating an interpolation curve using an interpolationmethod, such as the spline interpolation (Reference 1). The “splineinterpolation” is a method for interpolation used for generation ofinterpolation curves, finite element methods, function approximation,and extrapolation of experimental data, etc. (References 1 and 2).

Since a circadian expression curve obtained either by Fourier transformor by spline interpolation is expressed as a function of time,substitution of a certain time t into the function yields a value on thecircadian expression curve, and if a corresponding standard expressionlevel value is subtracted from the obtained value, followed by divisionby standard variation, a corresponding value on the relative expressioncurve can be calculated.

Estimation of internal biological time may be carried out as follows. Ina preliminary step, relative circadian expression curves are generatedfor each of 6 or more, preferably 30 or more, more preferably 50 ormore, particularly preferably 100 or more, arbitrary circadianoscillatory genes. The values, at time T, on these curves are comparedwith the relative expression levels of the corresponding circadianoscillatory gene products, which have been separately measured. The timeshowing the highest similarity in this comparison is estimated as theinternal biological time. For example, in the case where circadianexpression curves are generated in the form of cosine waves andsimilarity is calculated as the Pearson's product moment correlationcoefficient, if the highest similarity shows correlation coefficient of0.5 or higher, internal biological time can be estimated within an errorrange of 2 hours.

On the other hand, if sufficient similarity is not obtained between thetwo at all the times (e.g., Pearson's product moment correlationcoefficient<0.5), the presence of circadian rhythm disorders can besuggested. No particular limitation is imposed on the similarity, solong as it is a numeral value indicating similarity between an objectand another object. Preferably, Pearson's product moment correlationcoefficient is employed.

Circadian oscillatory genes used for estimating internal biological timemay be known circadian oscillatory genes or genes extracted by use ofthe DNA chip method or the like (including genes whose functions areunknown), so long as they exhibit characteristics of the circadianoscillatory genes as described in the present specification.

Other than the methods of estimating internal biological time bydefining similarity “a priori” as described above, internal biologicaltime can be estimated by use of a learning algorithm, specifically, witha support vector machine, genetic algorithm, neural network, etc., forimplementing similarity learning.

The following are references cited in the present specification:

-   Reference 1: “Numerical Recipes in C” William H. Press et al.,    Gijutsuhyoron-sha; Reference 2: “Spline function using C    parameters—Data Analysis, CG, and Differential Equation—” compiled    under supervision by Akira SAKURAI, authored by Keisuke SUGANO,    Kazumi YOSHIMURA, and Fumio TAKAYAMA, Tokyo Denki University Press;    Reference 3: “Cluster Analysis and its Application” translated and    supervised by Hideo NISHIDA, Uchida Rokakuho; and Reference 4:    “Fundamental Statistics I—an Introduction to the Statistics” edited    by The University of Tokyo, Komaba, College of Arts and Sciences    (Statistics), published by University of Tokyo Press.

INDUSTRIAL AVAILABILITY

As described above, according to the present invention, there isprovided internal biological time derivation means for obtaininginternal biological time information including judgment as to whether ornot a biological individual suffers a circadian rhythm disorder, andwhen the biological individual does not suffer a circadian rhythmdisorder, an estimated internal biological time of the biologicalindividual, through comparison between measurement data andabove-described circadian expression curves specified by theabove-described molecular timetables. Therefore, a comparison of assaydata on a specimen sampled at a single point in time with theabove-mentioned circadian expression curves specified by moleculartimetables generated for a predetermined portion of a predeterminedbiological species achieves, without need for sampling specimens aplurality of times, a simple judgment procedure for determining, on thebasis of a specimen sampled at a single point in time, as to thepresence or absence of a circadian rhythm disorder, and for estimatingthe internal biological time.

1. A molecular timetable generating apparatus for estimating internalbiological time of a biological individual on the basis of gene productquantity data as measured in standard specimens sampled fromindividuals, the apparatus being characterized by comprising: data inputmeans for inputting gene product quantity data of standard specimenseach sampled from a predetermined portion of each of a plurality ofindividuals of a predetermined biological species; circadian oscillatorygene selection means for selecting, from among the genes which areexpressed in the aforementioned standard specimens, circadianoscillatory genes whose time-course change in gene product quantity dataapproximates a cosine curve having a predetermined period; circadianexpression curve selection means for selecting, from among a pluralityof cosine curves having different phases and sharing a specific period,a circadian expression curve which is similar to the pattern oftime-course change in the expression product quantity of each of theabove-selected circadian oscillatory genes; and registration means forregistering information which identifies the selected circadianexpression curve.
 2. The molecular timetable generating apparatus asrecited in claim 1, characterized in that the registration means isconfigured so as to register, as a standard molecular time of thecircadian oscillatory gene, a point in time at which the circadianexpression curve reaches the maximum, in the molecular timetable usedfor estimating the internal biological time, and also to register, inthe molecular timetable, an average value and standard deviation, bothcalculated for each circadian oscillatory gene, of the expressionproduct quantity data, as a standard expression quantity and standardvariation of the circadian oscillatory gene, respectively.
 3. Aninternal biological time estimation apparatus for estimating internalbiological time of a biological individual on the basis of gene productquantity data as measured in specimens sampled from individuals, theapparatus comprising: molecular timetable storage means for storingmolecular timetables each of which specifies a circadian expressioncurve showing time-course change in the expression product quantity of acircadian oscillatory gene contained in a predetermined portion of apredetermined biological species; data input means for inputting thegene product quantity data of the circadian oscillatory genes containedin a specimen sampled from said predetermined portion of the biologicalindividual; and internal biological time derivation means for obtaininginternal biological time information including judgment as to whether ornot the biological individual suffers a circadian rhythm disorder, andwhen the biological individual does not suffer a circadian rhythmdisorder, an estimated internal biological time of the biologicalindividual, through comparison between the input measurement data andthe circadian expression curve specified by the molecular timetable. 4.A molecular timetable generating method which enables, by use ofinformation processing equipment, generation of a molecular timetablefor estimating internal biological time of a biological individual onthe basis of gene product quantity data as measured in standardspecimens sampled from individuals, the method comprising: a data inputprocedure for inputting gene product quantity data of standard specimenseach sampled from a predetermined portion of each of a plurality ofindividuals of a predetermined biological species; a circadianoscillatory gene selection procedure for selecting, from among the geneswhich are expressed in the aforementioned standard specimens, circadianoscillatory genes whose time-course change in gene product quantity dataapproximates a cosine curve having a predetermined period; a circadianexpression curve selection procedure for selecting, from among aplurality of cosine curves having different phases and sharing aspecific period, a circadian expression curve which is similar to thepattern of time-course change in the expression product quantity of eachof the above-selected circadian oscillatory genes; and a registrationprocedure for registering information which identifies the selectedcircadian expression curve.
 5. An internal biological time estimationmethod for estimating, by use of information processing equipment,internal biological time of a biological individual on the basis of geneproduct quantity data as measured in specimens sampled from individuals,the method being characterized by comprising: a molecular timetablestorage procedure for storing molecular timetables each of whichspecifies a circadian expression curve showing time-course change in theexpression product quantity of a circadian oscillatory gene contained ina predetermined portion of a predetermined biological species; a datainput procedure for inputting the gene product quantity data of thecircadian oscillatory genes contained in a specimen sampled from saidpredetermined portion of the biological individual; and an internalbiological time derivation procedure for obtaining internal biologicaltime information including judgment as to whether or not the biologicalindividual suffers a circadian rhythm disorder, and when the biologicalindividual does not suffer a circadian rhythm disorder, an estimatedinternal biological time of the biological individual, throughcomparison between the input measurement data and the circadianexpression curve specified by the molecular timetable.
 6. A moleculartimetable generating program which enables information processingequipment to perform: a data input procedure for inputting gene productquantity data of standard specimens each sampled from a predeterminedportion of each of a plurality of individuals of a predeterminedbiological species; a circadian oscillatory gene selection procedure forselecting, from among the genes which are expressed in theaforementioned standard specimens, circadian oscillatory genes whosetime-course change in gene product quantity data approximates a cosinecurve having a predetermined period; a circadian expression curveselection procedure for selecting, from among a plurality of cosinecurves having different phases and sharing a specific period, acircadian expression curve which is similar to the pattern oftime-course change in the expression product quantity of each of theabove-selected circadian oscillatory genes; and a registration procedurefor registering information which identifies the selected circadianexpression curve; said information processing equipment being providedfor generation of a molecular timetable for estimating internalbiological time of the biological individual on the basis of geneproduct quantity data as measured in standard specimens sampled fromindividuals.
 7. An internal biological time estimation program whichenables information processing equipment to perform: a moleculartimetable storage procedure for storing molecular timetables each ofwhich specifies a circadian expression curve showing time-course changein the expression product quantity of a circadian oscillatory genecontained in a predetermined portion of a predetermined biologicalspecies; a data input procedure for inputting the gene product quantitydata of the circadian oscillatory genes contained in a specimen sampledfrom said predetermined portion of the biological individual; and aninternal biological time derivation procedure for obtaining internalbiological time information including judgment as to whether or not thebiological individual suffers a circadian rhythm disorder, and when thebiological individual does not suffer a circadian rhythm disorder, anestimated internal biological time of the biological individual, throughcomparison between the input measurement data and the circadianexpression curve specified by the molecular timetable; said informationprocessing equipment being provided for estimating internal biologicaltime of the biological individual on the basis of gene product quantitydata as measured in standard specimens sampled from individuals.
 8. Aninternal biological time estimation system which enables generation of amolecular timetable for estimating internal biological time of abiological individual on the basis of gene product quantity data asmeasured in standard specimens sampled from individuals and estimationof internal biological time of the biological individual on the basis ofgene product quantity data as measured in a specimen sampled from thebiological individual; said system being characterized by comprising: aserver computer installed in an information center for providinginternal biological time information, and a terminal computer connectedto the server computer so as to transmit and receive informationtherebetween, wherein the server computer includes: standard data inputmeans for inputting gene product quantity data of standard specimenseach sampled from a predetermined portion of each of a plurality ofindividuals of a predetermined biological species; circadian oscillatorygene selection means for selecting, from among the genes which areexpressed in the aforementioned standard specimens, circadianoscillatory genes whose time-course change in gene product quantity dataapproximates a cosine curve having a predetermined period; circadianexpression curve selection means for selecting, from among a pluralityof cosine curves having different phases and sharing a specific period,a circadian expression curve which is similar to the pattern oftime-course change in the expression product quantity of each of theabove-selected circadian oscillatory genes; registration means forregistering, as a standard molecular time of the circadian oscillatorygene, a point in time at which the circadian expression curve reachesthe maximum, in the molecular timetable used for estimating the internalbiological time, and also for registering, in the molecular timetable,an average value and standard deviation, both calculated for eachcircadian oscillatory gene, of the expression product quantity data, asa standard expression quantity and standard variation of the circadianoscillatory gene, respectively; measurement data input means forinputting the gene product quantity data of the circadian oscillatorygenes contained in the specimen sampled from the said predeterminedportion of the biological individual; internal biological timederivation means for obtaining internal biological time informationincluding judgment as to whether or not the biological individualsuffers a circadian rhythm disorder, and when the biological individualdoes not suffer a circadian rhythm disorder, an estimated internalbiological time of the biological individual, through comparison betweenthe input measurement data and the circadian expression curve specifiedby the molecular timetable; and internal biological time transmissionmeans for transmitting the obtained internal biological time informationto the terminal computer.